Supplemental Information

Supplemental figure 1. Lists of whose expression was decreased (A) or increased (B) in the liver of Ire1α-null (Ire1Hepfe/-) mice, compared to the control (Ire1Hepfe/+) mice, in the absence

of TM treatment. The normalized average expression values were based on the microarray

analysis. The genes whose expression was changed at least 1.5-folds were listed (p0.05).

Supplemental figure 2. (A) Levels of intracellular triglycerides in liver tissues of Ire1α-null

(Ire1Hepfe/-) and control (Ire1Hepfe/+) mice after intraperitoneal injection of TM (2µg/gram body weight) or vehicle (150 μM dextrose) for 24 hours. The mice were fasted for 8 hours before euthanized for collecting samples. Each bar denotes the mean ± SEM (n=6 mice per group).

* P<0.05. (B-C) Levels of liver cellular phospholipids, including phosphatidylethanolamine (PtdEtn), phosphatidylcholine (PtdCho), and sphingomyelin (SM), in the Ire1Hepfe/- and Ire1Hepfe/+ mice after intraperitoneal injection with the vehicle or TM for 8 hours. Each bar denotes the mean ± SEM (n=3 mice per group). * P<0.05. (D) Western blot analysis of phoshocholine cytidylyltransferase alpha

(CCTα) in the Ire1Hepfe/- and Ire1Hepfe/+ mice after intraperitoneal injection with the vehicle

or TM for 8 hours. Levels of α-tubulin were determined as internal controls. The CCTα antibody

can detect an unspecific band below CCTα band with mouse liver tissue samples.

Supplemental figure 3. Quantitative real-time PCR analysis of the mouse Pparγ2 expression in the liver or fat tissue of mice after high-fat feeding or in the liver of mice after TM treatment. (A) Threshold cycle numbers of quantitative real-time PCR analysis for the Pparγ2

mRNA expression in the white fat and liver tissues of mice after the high-fat diet or in the liver

tissue of mice after TM treatment. C57BL/6J mice of 8-weeks old were fed with a high-fat diet (42%

1 fat, TD 88137, Harland Teklad) for 2 months. Total RNA was isolated from the white adipose or

liver tissue for the quantitative real-time PCR analysis. The Ire1α-null (Ire1αHepfe/-) and control

(Ire1αHepfe/+) mice were challenged with TM (2µg/gram body weight) or vehicle (150 µM dextrose)

for 8 hrs. Total RNA was isolated from the liver tissue for the quantitative real-time PCR analysis.

The real-time PCR primer sequences for amplifying the Pparγ2 mRNA are 5’-

TCTGGGAGATTCTCCTGTTGA-3’ and 5’-GGTGGGCCAGAATGGCATCT-3’. (B) The relative

Pparγ2 mRNA levels (fold changes) in the white adipose tissue (WAT) and liver of mice after the

high-fat diet for 2 months or in the liver of mice after TM treatment for 8 hrs. The fold changes of

the Pparγ2 mRNA levels were calculated based on the real-time PCR analysis (n=5 for high-fat-fed

mice group, and n=3 for TM-treated mice group).

Supplemental figure 4. Quantitative real-time RT-PCR analysis for fold changes in expression of

the Acox1, Cpt1a, Cyp4a10, Fabp1, MTTP, Fatp2, Fsp27, aP2, and Fat/Cd36 mRNAs in the livers of the Ire1Hepfe/- and Ire1Hepfe/+ mice. Total RNAs from the livers of mice challenged with TM (2

µg/gram body weight) or vehicle for 8 hours were subjected to quantitative real-time RT-PCR analysis. Expression values were normalized to the β-actin mRNA levels. Fold changes of mRNA levels were shown by comparison to the expression level of mRNA in one of the control

(Ire1Hepfe/+) mice. Each bar denotes the mean ± SEM (n=6 mice per group). * P<0.05; ** P<0.01.

Supplemental figure 5. Quantitative real-time RT-PCR analysis for fold changes in expression of the Pparα, Pparδ, Pgc-1α, Pgc-1β, Fasn, and Srebp-1c mRNAs in the livers of the Ire1Hepfe/- and

Ire1Hepfe/+ mice. Total RNAs from the livers of mice challenged with TM (2 µg/gram body weight) or

vehicle for 8 hours were subjected to quantitative real-time RT-PCR analysis. Expression values

were normalized to the β-actin mRNA levels. Fold changes of mRNA levels were shown by

2 comparison to the expression level of mRNA in one of the control (Ire1Hepfe/+) mice. Each bar denotes the mean ± SEM (n=6 mice per group). * P<0.05.

Supplemental figure 6. Mice with hepatocyte-specific Ire1α deletion exhibit increased apoptosis in response to Bortezomib treatment. Ire1α-null (Ire1Hepfe/-) and control (Ire1Hepfe/+) mice were injected with Bortezomib (1µg/gram body weight) into the tail vein. At 36 hours after injection, the mice were fasted for 8 hours prior to euthanasia and collection of liver tissue samples. (A)

Immunofluorescent TUNEL staining of liver tissue sections for analysis of DNA fragmentation

(magnification: 400×). The apoptotic cells exhibited green fluorescence. The cells were also stained with propidium iodide (PI), to identify all cells by strong red cytoplasmic fluorescence. (B)

The percentages of TUNEL-positive cells (green fluorescence) determined by calculating the number of TUNEL-positive cells divided by the number of PI-positive cells (red fluorescence) from five fields of each slide. ** P<0.01.

Supplemental figure 7. Ire1α-null mice displayed profound hepatic steatosis after partial hepatectomy. Ire1α-null (Ire1Hepfe/-) and control (Ire1Hepfe/+) mice of 3-months old were subjected

to 2/3 partial hepatectomy as previously described (Mitchell & Willenbring, 2008). Briefly, the mice

were anesthetized with 2% isoflurane and 2 liters/min oxygen flow for 25g body weight, followed by

removal of 2/3 liver lobes. At 60 hrs after the hepatectomy surgery, the liver tissues were collected

for lipid droplet staining and measurement of hepatic triglycerides. (A) Liver tissue sections from

the Ire1α-null (Ire1Hepfe/-) and control (Ire1Hepfe/+) mice at 60 hrs after hepatectomy were stained with Hematoxylin and Eosin (Magnification: 200x). Lipid droplets were shown by white droplets in the Hematoxylin and Eosin-stained tissue sections. PH, partial hepatectomy. (B) Levels of

intracellular triglycerides in the livers of Ire1α-null and control mice under the non-stressed

3 condition or after 60 hrs of partial hepatectomy. Measurement of intracellular triglyceride levels was performed using a triglyceride assay kit according to manufacturer’s instruction (BioAssay

Systems, ETGA-200). ** P<0.01, n=3 mice per group.

Supplemental figure 8. A model depicting the role of IRE1α in maintaining hepatic lipid homeostasis.

References

Mitchell C, Willenbring H (2008) A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat Protoc 3: 1167-1170

4 Supplemental figure 1A

64 genes whose expression was decreased in the knockout (KO) liver (at least 1.5-fold decrease compared to that in the control (Ctl) liver, p<0.05)

Ctl KO KO/Ctl Gene Symbol Genbank N.A. value N.A. value Folds Gene Description Snrpn BB709603 1.106336 0.1591251 6.953 small nuclear ribonucleoprotein N Tmepai AV291712 1.256455 0.1946048 6.456 transmembrane, prostate induced RNA Xlr3a NM_011726 0.883916 0.1382069 6.396 X‐linked lymphocyte‐regulated 3A Pola2 AV326590 0.834934 0.1744544 4.786 (DNA directed), alpha 2 Arf4 BB754834 1.003703 0.2105785 4.766 ADP‐ribosylation factor 4 Zfp312 NM_080433 0.596788 0.1560784 3.824 zinc finger protein 312 Syt6 AV165454 0.964552 0.2616363 3.687 VI Cyb561 BC006732 1.217992 0.34914 3.489 cytochrome b‐561 Aff4 BB233318 0.850025 0.2504085 3.395 AF4/FMR2 family, member 4 Twist2 NM_007855 0.768495 0.2327472 3.302 twist homolog 2 (Drosophila) Abi1 AW912678 0.457666 0.143734 3.184 abl‐interactor 1 Lrrcc1 AK004646 1.040055 0.3363853 3.092 leucine rich repeat and coiled‐coil domain containing 1 Dnajb5 AI664344 0.626192 0.2106713 2.972 DnaJ (Hsp40) homolog, subfamily B, member 5 Air BB365661 1.482377 0.6372369 2.326 antisense Igf2r RNA Slc1a4 BB277461 0.748341 0.3258698 2.296 solute carrier family 1, member 4 Rdh11 AB030503 1.864846 0.8584489 2.172 retinol dehydrogenase 11 Ccl9 AF128196 0.508151 0.2365402 2.148 chemokine (C‐C motif) ligand 9 V2R2 NM_019918 0.892602 0.4267216 2.092 vomeronasal neurons putative pheromone receptor V2R2 LOC433755 AA189214 0.646608 0.3145227 2.056 hypothetical LOC433755 Mvd NM_138656 0.830283 0.4144092 2.004 mevalonate (diphospho) decarboxylase Crim1 BM234411 0.995102 0.5075338 1.961 Cysteine‐rich repeat‐containing protein CRIM1 (Crim1) Trpm3 BB125842 1.006872 0.5174327 1.946 transient receptor potential cation channel, subfamily M, member 3 Cxadr BE824924 0.673235 0.3464771 1.943 coxsackievirus and adenovirus receptor Mvk BC005606 2.504142 1.3035088 1.921 mevalonate Arrdc1 BC018501 1.312878 0.6950923 1.889 arrestin domain containing 1 Pstpip2 BC002123 1.312671 0.7104448 1.848 proline‐serine‐threonine phosphatase‐interacting protein 2 Tlk2 AK014829 0.649527 0.3603371 1.803 tousled‐like kinase 2 (Arabidopsis) Dnajc3 BE624323 0.54686 0.3051605 1.792 DnaJ (Hsp40) homolog, subfamily C, member 3 Spcs2 BI078449 1.131494 0.645468 1.753 signal peptidase complex subunit 2 homolog Nck1 BB296097 1.236411 0.7083386 1.746 non‐catalytic region of adaptor protein 1 Tbl2 NM_013763 1.15668 0.666081 1.737 transducin (beta)‐like 2 Sc4mol AK005441 2.50624 1.4444478 1.735 sterol‐C4‐methyl oxidase‐like Acsl3 AK012088 1.310414 0.755519 1.734 acyl‐CoA synthetase long‐chain family member 3 Fdft1 NM_010191 1.928618 1.1140472 1.731 farnesyl diphosphate farnesyl 1 Ssr4 NM_009279 1.220169 0.7124034 1.713 signal sequence receptor, delta Smyd5 BF160651 0.687019 0.402269 1.708 SET and MYND domain containing 5 Dntt AF316014 1.230267 0.7333166 1.678 deoxynucleotidyltransferase, terminal Mall BC012256 0.961877 0.5742328 1.675 mal, T‐cell differentiation protein‐like Brwd3 BG066622 1.797896 1.077777 1.668 bromodomain and WD repeat domain containing 3 Gnpnat1 AK008566 0.759661 0.4595889 1.653 glucosamine‐phosphate N‐acetyltransferase 1 Pip5k1b BB822856 0.706625 0.4305397 1.641 phosphatidylinositol‐4‐phosphate 5‐kinase, type 1 beta Letm1 BG060855 1.623128 0.9922701 1.636 leucine zipper‐EF‐hand containing transmembrane protein 1 Atf2 BM119623 0.951484 0.581706 1.636 activating transcription factor 2 Mknk1 AK010697 0.762839 0.4678569 1.63 MAP kinase‐interacting serine/threonine kinase 1 Mvd NM_138656 0.906597 0.556162 1.63 mevalonate (diphospho) decarboxylase Tead1 BB546942 1.416145 0.8726368 1.623 TEA domain family member 1 Btg2 NM_007570 0.561845 0.3490928 1.609 B‐cell translocation gene 2, anti‐proliferative Ythdf1 BB350365 1.183 0.738022 1.603 YTH domain family 1 Atp11c AI593492 2.060446 1.2864633 1.602 Atpase, class VI, type 11C Gorasp2 AK010664 1.160286 0.7266653 1.597 golgi reassembly stacking protein 2 Dnajb9 NM_013760 0.761702 0.4798189 1.587 DnaJ (Hsp40) homolog, subfamily B, member 9 Tmed3 NM_025360 0.711924 0.4486596 1.587 transmembrane emp24 domain containing 3 Mrgpre BB373312 1.614723 1.0224634 1.579 MAS‐related GPR, member E Steap2 BB529332 1.576645 1.0122856 1.558 six transmembrane epithelial antigen of prostate 2 Gpr24 BE647763 1.959893 1.2711265 1.542 G protein coupled receptor 24 Nudcd1 AW542236 1.036232 0.6726758 1.54 NudC domain containing 1 H47 AK005204 0.669161 0.4350573 1.538 histocompatibility 47 Ropn1l AF305427 1.983781 1.2916508 1.536 ropporin 1‐like Rnu22 BQ177137 0.328328 0.215546 1.523 RNA, U22 small nucleolar Rnu22 BQ177137 0.316196 0.2075945 1.523 RNA, U22 small nucleolar Gnal BB306011 1.540748 1.0144672 1.519 guanine nucleotide binding protein, alpha stimulating, olfactory type Ikbke NM_019777 0.975424 0.6426376 1.518 inhibitor of kappaB kinase epsilon Acss2 NM_019811 1.916789 1.2638172 1.517 acyl‐CoA synthetase short‐chain family member 2 Stch BE650268 0.992851 0.6611507 1.502 stress 70 protein chaperone, microsome‐associated, human homolog

N.A. value: normalized average exppression value KO/Ctl: ratios of gene expression values of IRE1α null vs control. Supplemental figure 1B

57 genes whose expression was increased in the knockout (KO) liver (at least 1.5-fold increase compared to that in the control (Ctl) liver, p<0.05)

Ctl KO KO/Ctl Gene Symbol Genbank N.A. value N.A. value Folds Description Atp2a2 BG063183 0.515533 0.7786049 0.662 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 Cdon BB125469 0.628787 0.9558229 0.658 Adhesion molecule‐related/down‐regulated by oncogenes Rnu22 AW552536 0.412008 0.6265222 0.658 RNA, U22 small nucleolar Gm967 AW457804 0.54835 0.8395036 0.653 gene model 967, (NCBI) Ddit3 NM_007837 0.028109 0.0433388 0.649 DNA‐damage inducible transcript 3 Zfp207 AI646720 0.37787 0.5858806 0.645 Zinc finger protein 207, mRNA Nfasc BB409198 0.989849 1.5413092 0.642 neurofascin Cad AK010453 0.334711 0.5245007 0.638 carbamoyl‐phosphate synthetase 2 Zfp592 BB478535 0.619713 0.9814639 0.631 zinc finger protein 592 4930432B04RikBB437636 0.673198 1.0686985 0.63 Cytoskeleton associated protein 5, mRNA Rapgef2 BM120546 0.513634 0.8223091 0.625 Rap guanine nucleotide exchange factor (GEF) 2, mRNA Pbx3 BB554874 0.575067 0.9251468 0.622 pre B‐cell leukemia transcription factor 3 Nedd4l BB360028 0.25571 0.4139056 0.618 Neural cell developmentally down‐regulated gene 4‐like Apoa5 AA254866 1.401669 2.2762778 0.616 Apolipoprotein A‐V (Apoa5), mRNA Rab2b AK005230 0.506506 0.8229689 0.615 RAB2B, member RAS oncogene family Mpp4 AB059357 1.238018 2.0285037 0.61 , palmitoylated 4 Phr1 BM238052 0.589302 0.9722936 0.606 Pam, highwire, rpm 1 (Phr1), mRNA Mettl4 BB701076 0.506154 0.8473486 0.597 methyltransferase like 4 Dock4 BB656539 1.514044 2.5413232 0.596 dedicator of cytokinesis 4 Phip BM218981 1.295397 2.1799352 0.594 pleckstrin homology domain interacting protein Kctd13 BQ177107 0.543341 0.9328961 0.582 potassium channel tetramerisation domain containing 13 Pkp2 AK004731 0.513869 0.8937289 0.575 plakophilin 2 Hist1h1c BB533903 0.315064 0.5486828 0.574 histone 1, H1c Bach2 BI104953 1.052494 1.8663629 0.564 BTB and CNC homology 2 (Bach2), mRNA Arfrp2 AI785253 0.434556 0.7710369 0.564 ADP‐ribosylation factor related protein 2 (Arfrp2), mRNA Gle1l AV228343 0.422612 0.7501699 0.563 GLE1 RNA export mediator‐like Derl3 AK007348 0.019531 0.0357669 0.546 Der1‐like domain family, member 3 Mdm1 AV313203 0.365482 0.6745294 0.542 transformed mouse 3T3 cell double minute 1 Bcl7c BB176719 0.964105 1.8177955 0.53 B‐cell CLL/lymphoma 7C Yipf4 AV216410 0.507096 0.9613949 0.527 Yip1 domain family, member 4 Eif3s6ip AV216873 0.648856 1.2522556 0.518 translation initiation factor 3, subunit 6 interacting protein Tnfrsf9 BC028507 0.611113 1.1897492 0.514 tumor necrosis factor receptor superfamily, member 9 Arfrp2 BM214060 0.427078 0.8488674 0.503 ADP‐ribosylation factor related protein 2 (Arfrp2), mRNA Nedd4l BB309512 0.321554 0.64352 0.5 neural cell developmentally down‐regulated gene 4‐like Ddit3 BB200603 0.349908 0.7010221 0.499 DNA‐damage inducible transcript 3 Nr4a2 NM_013613 0.468023 0.9377375 0.499 nuclear receptor subfamily 4, group A, member 2 Mnab BM210927 0.471156 0.9532796 0.494 membrane associated DNA binding protein Col9a3 BG074456 0.775138 1.606882 0.482 procollagen, type IX, alpha 3 Phf6 BF147362 0.449741 0.9673274 0.465 PHD finger protein 6 Cdkn1a AK007630 0.085497 0.1869583 0.457 cyclin‐dependent kinase inhibitor 1A (P21) Ddit4 AK017926 0.262658 0.5748734 0.457 DNA‐damage‐inducible transcript 4 Psat1 BC004827 0.012318 0.0273046 0.451 phosphoserine aminotransferase 1 Susd4 BF455403 1.061193 2.371801 0.447 sushi domain containing 4 Tktl1 C79967 0.777513 1.8779701 0.414 Transketolase‐like 1 (Tktl1), mRNA Phox2a NM_008887 0.347061 0.8547047 0.406 paired‐like homeobox 2a Styk1 AV375472 0.228523 0.5910682 0.387 serine/threonine/tyrosine kinase 1 Esrrg BM120183 0.663688 1.7384369 0.382 ‐related receptor gamma Scara5 BC016096 0.888253 2.352793 0.378 scavenger receptor class A, member 5 (putative) Mrvi1 U63408 0.38448 1.1671803 0.329 MRV integration site 1 Ddx50 AA518255 0.140597 0.4892876 0.287 DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 50 H1fx BI416101 0.128748 0.4861345 0.265 H1 histone family, member X Bicc1 BB497148 0.161315 0.6333181 0.255 Bicaudal C homolog 1 (Drosophila) (Bicc1), mRNA Asns BC005552 0.121902 0.5628594 0.217 asparagine synthetase Trpc1 BB817939 0.176141 1.1086509 0.159 transient receptor cation channel, subfamily C, member 1 Mrc2 AW555385 0.132763 0.9969737 0.133 Mannose receptor, C type 2 (Mrc2), mRNA Asns AV212753 0.066149 0.5234336 0.126 asparagine synthetase Atad1 AV017609 0.130715 1.0529606 0.124 ATPase family, AAA domain containing 1

N.A. value: normalized average exppression value KO/Ctl: ratios of gene expression values of IRE1α null vs control. A220 B C 200 ** 180 Vehicle TM HepFe/+ 160 HepFe/- HepFe/+ HepFe/+ 140 HepFe/- HepFe/- 120 100 80 60 g Lipid/mg liver g Lipid/mg

 40 g Lipid/mg liver g Lipid/mg liver  20  0 Vehicle TM PtdEtn PtdCho SM PtdEtn PtdCho SM

D Vehicle TM HepFe/+ HepFe/- HepFe/+ HepFe/- 1 2 3 1 2 3 1 2 3 1 2 3 CCT

tubulin

Supplemental figure 2 A B Threshold cycle ieI#Co itHg a itCo itHg a itCo itHg a itCo itHighFatDiet mice ID# Chow Diet HighFatDiet ChowDiet HighFatDiet ChowDiet HighFatDiet ChowDiet mice ID# 3 2 1 5 4 3 2 1 aietncmcnsln uiayi aietncmcnsln tunicamycin saline tunicamycin saline tunicamycin saline tunicamycin saline 19 19 / 20 52 56 53 14.11 15.41 15.71 15.30 15.70 14.66 15.61 14.84 14.67 14.84 14.81 15.27 16.20 15.02 15.70 15.70 16.19 32.02 15.47 N/A 31.42 19.36 19.34 N/A 18.75 33.33 20.73 33.34 20.64 31.97 19.30 32.97 24.82 32.38 25.32 29.20 31.99 31.89 25.32 33.07 24.37 25.38 28.43 28.38 29.03 32.82 34.95 33.12 / 80 76 54 90 90 56 15.30 15.32 15.66 15.78 19.03 19.75 19.03 19.30 25.41 26.96 27.68 27.35 28.01 30.73 N/A N/A wild-type Liver Relative PPAR2 mRNA levels 10 15 20 0 1 2 5 PPARgamma2 PPARgamma2 p<0.01 liver Chow Diet p<0.01

High Fat Diet White adipose IRE-KO

WAT Chow Diet

High Fat Diet

saline C57BL/6J Wild-type Tunicamycin IRE KO liver wild-type Liver saline Supplemental figure3

Tunicamycin beta-actin beta-actin White adipose IRE-KO A 4 ** CTL 3.5 IRE1α null 3 * CTL-TM 2.5 ** IRE1α null-TM 2 1.5 1 0.5 0 Acox1 Cpt1a Cyp4a10 B 3.5 CTL IRE1α null 3 ** CTL-TM 2.5 * IRE1α null-TM 2 * 1.5

1

0.5

0 Fabp1 MTTP Fatp2 C 3 * * CTL IRE1α null 2.5 * CTL-TM 2 IRE1α null-TM

1.5

1

0.5

0 Fsp27 aP2 Fat/Cd36

Supplemental figure 4 1.5

* HepFe/+ (Vehicle) 1.2 HepFe/- (Vehicle) HepFe/+ (TM) HepFe/- (TM) 0.9

0.6

0.3 mRNA levels (fold changes) (fold levels mRNA 0 Ppara Ppard Pgc-1a Pgc-1b Fasn Srebp-1c

Supplemental figure 5 A B

HepFe/+ (BTZ) HepFe/- (BTZ) 25 **

20

15

10

5 TUNEL positive (%) TUNEL

0 Hep Hep Fe/+ Fe/-

Supplemental figure 6 A

Hep Fe/+ PH Hep Fe/- PH

B ** 100

75

50 g/mg) 

25

0 Triglyceride/liver protein Triglyceride/liver ( HepFe/+ HepFe/- HepFe/+ HepFe/-

Afrer hepatectomy

Supplemental figure 7 ER stress

IRE1

Secretion: C/EBP/ apolipoprotein

PPAR Plasma lipids

Lipogenesis, steatosis

Supplemental figure 8